Discovery of Tick-Borne Karshi Virus Implies Misinterpretation of the Tick-Borne Encephalitis Virus Seroprevalence in Northwest China

Abstract

Despite few human cases of tick-borne encephalitis virus (TBEV), high rates of TBEV seroprevalence were reported among humans and animals in Xinjiang Uygur Autonomous Region in Northwestern China. In this study, the Karshi virus (KSIV) was identified and isolated from Hyalomma asiaticum ticks in Xinjiang. It belongs to the genus Flavivirus of the family Flaviviridae and is closely related to TBEV. KSIV infects cell lines from humans, other mammals and ticks, and causes encephalitis in suckling mice. High minimum infection rates (4.96%) with KSIV were detected among tick groups. KSIV infections have occurred in sheep and marmots, resulting in antibody-positive rates of 2.43 and 2.56%, respectively. We further found that, of the KSIV antibody-positive serum samples from animals, 13.9% had TBEV exposure showing cross-reaction to KSIV, and 11.1% had KSIV infection resulting in cross-reaction to TBEV; 8.3% were likely to have co-exposure to both viruses (or may be infected with one of them and present cross-reactivity with the other). The results revealed a substantial KSIV prevalence among ticks in Xinjiang, indicating exposure of animals to KSIV and TBEV. The findings implied misinterpretation of the high rates of TBEV seroprevalence among humans and animals in previous studies. There is a need to develop detection methods to distinguish KSIV from TBEV and to perform an in-depth investigation of KSIV and TBEV prevalence and incidence in Northwestern China, which would enhance our preparation to provide medical treatment of emerging diseases caused by tick-borne viral pathogens such as KSIV.

Keywords: Karshi virus; cross-reaction; prevalence; serological correlation; tick-borne encephalitis virus.

FIGURE 1

Karshi virus (KSIV) isolation, characteristics and analysis of viral particles. (A) Immunofluorescence assays to survey KSIV isolation in BHK-21 cells. The images were taken from different passages showing KSIV proliferation. Cells were immunostained and green fluorescence indicates the cells infected by KSIV. P1, the first passage; P2, the second passage; P3, the third passage; P4, the fourth passage. (B) Plaque assay to verify KSIV isolation in BHK-21 cells with the P4 supernatants. Cells were stained with crystal violet to show plaques at 3 days post-infection. (C) TEM analysis of KSIV particles purified from supernatants harvested from BHK-21 cells infected with KSIV at 3 days post-infection. The clarified supernatants were subjected to ultracentrifugation. The fractions containing viral particles were harvested and used in negative-staining. (D) The image obtained by TEM shows that virus particles were located in the cytoplasm of infected BHK-21 cells. The enlarged area on the right shows groups of virus particles (arrow). N, nucleus; C, cytoplasm; NM, nuclear membrane.

FIGURE 2

The susceptibility of cell lines derived from different hosts to KSIV and viral one-step growth curve test. (A) Cell lines derived from humans (SH-SY5Y, U-87 MG, SW-13, HEK239 and HepG2), other mammals (Vero, BHK-21, MDBK, MDOK, and DH82), mosquito (C6/36) and ticks (HAE/CTVM9 and IDE8) were infected with KSIV at MOI of 1. The cells were cultured for 48 h and examined by immunofluorescence for KSIV E protein. KSIV infection was detected in all cell lines except MDBK, HAE/CTVM9 and C6/36. (B) The viral one-step growth curve in various cell lines. BHK-21 cells were infected with KSIV at an MOI of 5 and the supernatant for collected infection of cell lines of different species: human (SH-SY5Y, SW13) and hamster (BHK-21), virus titers were measured by end-point dilution assays.

FIGURE 3

The pathogenicity of KSIV infection in 2- and 9-day-old suckling C57BL/6 mice. The 2- and 9-day-old suckling C57BL/6 mice (2- and 9-dM) were inoculated with KSIV intracranially (1 × 10⁴ PFU) and intraperitoneally (2 × 10⁴ PFU) and monitored for clinical symptoms and mortality over 15 days. (A) Survival analysis of mice infected with KSIV. (B) Bodyweight changes of mice infected with KSIV. Mice were inoculated with KSIV and different tissues were collected at 6- and 8-days post inoculation (n = 3/group) to enable comparisons of virus loads between the 2-day-old (C) and the 9-day-old (D) infected mice groups. For each time point, the measured values are the average of three mice. Error bars represent standard deviations. Data shown are pooled from three independent experiments. n.s. indicates no significant difference, *p < 0.05, ^**p < 0.01. (E) H&E staining of brains from 2- to 9-day-old KSIV-infected and uninfected suckling mice. At 6- and 8-days post inoculation, mice were euthanized, and brains were H&E stained. Representative images of the brain from (a) thalamus, (b) prosencephalon, and (c) hippocampus. Scale bars represent 100 μm. The enlarged images of interest show details demonstrating inflammation and neuronal necrosis. (F) Double immunofluorescence staining was performed on mouse brain sections from 2- to 9-day-old mice 8 days after KSIV infection, and on uninfected mice. The mature neurons were marked with red fluorescence (Alexa Fluor 555), KSIV antigens were stained with green fluorescence (Alexa Fluor 488), and the nuclei were stained with Hoechst 33258.

FIGURE 4

Map of Xingjiang Region showing locations where KSIV infection was detected. Molecular, epidemiological, and serological investigations were conducted on ticks and field-collected animal sera from Xinjiang, revealing that the animals had serological exposure to tick-borne encephalitis virus (TBEV) as well as to KSIV. The map of Xinjiang, China, shows tick sampling locations and molecular epidemiology of KSIV, and anti-KSIV and anti-TBEV antibody detection in animal sera. The silhouette of a tick with a red background represents a sampling location where KSIV nucleic acid was detected, and the silhouette of a transparent tick represents a sampling location where KSIV was not detected. The silhouettes of sheep and rats on a yellow background represent the sampling locations (black dots) where KSIV-antibodies were detected, while the locations where the animal serum samples also had neutralizing antibodies to TBEV are indicated by red dots. Gray stippling indicates arid desert areas. S-Usu, South of Usu City; N-Usu, North of Usu City; S-Jinghe, South area of Jinghe County; N-Jinghe, North of Jinghe County.

FIGURE 5

Karshi virus has serological cross-reactivity with TBEV. The determination of KSIV and TBEV serological cross-reaction and neutralization was based on the immunofluorescence test. Percentages of KSIV-infected/TBEV-infected cells were obtained and normalized with the Operetta High-Content Imaging System (PerkinElmer). KSIV and TBEV serological cross-reaction test. The efficiencies of the lab-prepared polyclonal antibodiesα-KSIV-E (A) and α-TBEV-E (B) reacting to the KSIV- and TBEV-infected cells were evaluated using GraphPad Prism software (Version 8) least square fitting method to determine the detection efficiency curve. The KSIV-infected mouse serum dilution was determined as an antibody titer that inhibited 50% (C) and 90% (D) of KSIV and TBEV infection. The TBEV-infected mouse serum dilution was determined as an antibody titer that inhibited 50% (E) and 90% (F) of KSIV and TBEV infection. The data were shown as means ± SD. ^**p < 0.01, ^***p < 0.001.